Acute respiratory infection is now the leading cause of mortality in young children under 5 years of age, accounting for nearly one fifth (20%) of childhood deaths worldwide, and killing 2-3 million children each year. Human parainfluenza viruses and respiratory syncytial virus (RSV) cause the majority of childhood croup, bronchiolitis and pneumonia in the U.S., yet no drugs or vaccines are available. Development of antiviral drugs for these viruses has been a great challenge in the field because of gaps in fundamental knowledge about these viruses. We propose to apply fundamental research results in order to develop a new antiviral strategy based on inhibiting fusion during viral entry. Fusion inhibitory peptides can block viral fusion intermediates to prevent entry and infection. We have shown that the efficacy of peptide inhibitors for paramyxoviruses (such as parainfluenza and RSV) depends on three variables: (i) Strength of interaction of the peptide with the target fusion protein;(ii) Time window of access to the target sequence;(iii) Location of the peptide in proximity to the target fusion protein. We propose to use this new information to develop highly effective peptide fusion antivirals that inhibit both of these two important pediatric respiratory pathogens;to investigate the mechanisms of resistance to fusion inhibitors so as to avoid resistance;and to test these hypotheses in a valid animal model of disease. We will thus establish the in vivo potential of these fusion inhibitors. (1) Design and testing of anti-parainfluenza and anti-respiratory syncytial virus fusion inhibitory peptides targeted to the plasma membrane where fusion occurs. (a) Biophysical data and crystal structure analysis will be used to enhance peptide inhibitor binding to F. (b) Effective peptides will be targeted to the plasma membrane where fusion occurs, increasing their access to the F heptad repeat region, in order to enhance their action. We will determine whether the increased efficacy that we find by adding a cholesterol group to peptides is due to a generic increase of the effective concentration at the membrane surface, and/or a specific enrichment in lipid rafts, where virus entry occurs. (2) Determinants of viral resistance: Mechanisms and strategies for avoiding resistance. We will assess the determinants of resistance to membrane-anchored highly effective peptide inhibitors, and whether selection for resistance is reduced by targeting with peptides an earlier stage of F-activation, or by increasing the concentration of inhibitor at the location of receptor binding. (3) In vivo efficacy of modified peptide inhibitors. Effective peptides will be tested in vivo in the cotton rat, to establish the desirability of developing these inhibitors as clinically useful antiviral agents. Effective therapy for pediatric respiratory viruses would decrease the cost of health care for children in the U.S. and would significantly impact child health. PUBLIC HEALTH RELEVANCE: Acute respiratory infection is now the leading cause of mortality in young children under 5 years of age, accounting for nearly one fifth (20%) of childhood deaths worldwide, and killing 2-3 million children each year. Human parainfluenza viruses and respiratory syncytial virus cause the majority of childhood croup, bronchiolitis and pneumonia in the U.S. Despite the huge impact of these diseases on illness and hospitalization of young infants worldwide, no drugs or vaccines are available. Development of antiviral drugs for these viruses has been a great challenge in the field because of gaps in fundamental knowledge about these viruses. We propose to apply the results of fundamental research to develop a new antiviral strategy based on inhibiting fusion during viral entry. Effective therapy for pediatric respiratory viruses would significantly decrease the cost of health care for children in the U.S. and tremendously impact child health.